The project initially focused on an all-trans retinoic acid (atRA)-regulated orphan nuclear receptor TR2 in atRA signal transduction in development and embryonic stem cell (ESC). The long-term goal is to understand signaling pathways of atRA particularly in regulating stem cell property. The previous cycle (5/1/2012- 4/30/2016) aimed to examine 1) a then unknown non-canonical activity of atRA and its mediator and 2) atRA-elicited chromatin remodeling of stemness loci Oct-4/Nanog in ESC. Both aims were accomplished with 13 papers directly resulted from this support and 19 additional papers partially supported by this project. Four major accomplishments are: 1) establishing atRA's non-canonical (not mediated by RA receptors [RARs]) activation of ERK1/2 that stimulates post-translational modifications (PTMs) of proteins regulating chromatin remodelers and contributing to ESC genome organization, 2) identifying cellular retinoic acid binding protein 1 (Crabp1) as the mediator of this newly established non-canonical activity of atRA, 3) generating Crabp1 knockout (Crabp1KO) ESC and mice in C57/BL6 background and uncovering phenotype including expanded Neural Progenitor Cell (NPC) population in hippocampus, improved memory function and extended ESC cell cycle G1 phase, 4) identifying Crabp1-selective compounds C3 and C4 that activate Crabp1 without involving RARs, which provides tools to specifically examine non-canonical atRA signaling. With these, it is hypothesized that Crabp1-mediated non-canonical activity of atRA alters ERK1/2 activity to initiate regulatory proteins' PTMs for genome organization, followed by canonical atRA signaling that activates target gene transcription, ultimately the two signaling pathways effectively commits ESC into differentiation. The immediate goal is to comprehensively study non-canonical, Crabp1-mediated atRA signaling and understand integration of non-canonical and canonical signaling in the context of ESC genome maintenance (in vitro) and NPC homeostasis and brain functions (in vivo). Aim 1: To dissect molecular mechanisms of Crabp1-mediated non-canonical atRA signaling pathway by examining i) how holo-Crabp1 activates ERK1/2, ii) molecular features of atRA- vs. C3/C4-Crabp1 signaling complexes (signalsome), and iii) substrates of atRA-Crabp1 activated ERK1/2 as compared to that of canonical growth factor-activated ERK1/2 in ESC and NPC. Aim 2: To understand integration of canonical (RAR-mediated) and non- canonical (Crabp1-mediated) atRA signaling in the context of ESC genome maintenance and differentiation into NPC, and manifestation in modulating brain function such as memory by examining i) non-canonical activity of atRA (using C3/C4) in organizing topologically associated domain (TAD) for stemness loci in WT and Crabp1KO ESC/NPC, 2) effects on chromatin remodeling complexes Brg/Brm (ESC) and LSD1 (for NPC) in WT and Crabp1KO, and 3) integration of canonical and non-canonical pathways in regulating NPC homeostasis and hippocampus-dependent memory in animals.